During the past 100 years, cell cultures have resulted in many applications in the field of biotechnology. Progress in cell culture, especially in obtaining higher cell productivity, has allowed the development of new processes for production of recombinant proteins and vaccines, biomass and new uses of cells such as cell therapies.
Cells are mainly cultured for two applications. Firstly, cell amplification by subculture will result in increases in biomass production of viable cells. These viable cells can be used for such as cell therapies, for viral infections and to obtain infected cells and the like. The second application is in producing and isolating biological compounds of interest typically, but not always, present inside the cells, such as polynucleotides, proteins, animal pathogens, or fragments thereof. These bioproducts can also be incorporated into other products like DNA vaccines, subunit vaccines, viral vaccines, gene therapy compositions, drugs and the like.
A major problem of the cell culture is the formation of cell aggregates formed during the culture. Cell aggregates, by limiting access of the cells to nutrients and by contact growth inhibition, reduces the culture yield in terms of biomass production and of compounds of interest. In addition, cell aggregation increases cell death, primarily due to apoptosis. For biomass production harvested cells must not be dead or dying to provide for optimal subculturing.
Cells grow either attached to a surface (i.e. anchorage dependent) or in suspension (i.e. anchorage independent). Most cultures of animal cells are anchorage dependent and grow in single-cell layers (monolayers) or on the surface of micro-carriers, in dishes or flasks. Roller bottle technology was developed for cultivating larger number of anchorage-dependent animal cells (Gey G. O., Am. J. Cancer, 17: 752-756 (1933)) although a later improvement came from the use of micro-carriers in bioreactors, which permits an increase in the available growth area for cells per unit of volume (van Wezel A. L., Nature, 216: 64-65 (1967)).
These technologies have now been used for more than 20 years in the pharmaceutical and medical fields for processes such as cell growth and infection, vaccine preparation, recombinant protein expression, and plant cell cultivation. Many of these techniques have been published and are routinely used (See for example Freshney, R. I. Culture of animal cells: a manual of basic techniques: 3rd edition 1994).
Typically during culture of anchorage-dependent cells, when the culture reaches confluency, it is desirable to disaggregate the culture into individual cells that retain viability. Cultures of anchorage-independent cells also exhibit cell clusters, and that the problem of cell cluster dispersion exists, irrespective of what type of anchorage the cells have. The resulting disaggregated suspension can then be subcultured or be used directly as a source of a pharmaceutically acceptable compound. Dispersion of cells can be a solution to the inherent problems of cell aggregation but is also problematic, however, due to the fragility of cells resulting in stresses and deaths.
Cells are often so well attached to the underlying culture vessel surface that proteolytic enzymes (such as trypsin, collagenase, pronase), chelating agents (such as ethylenediaminetetraacetic acid) and mechanical forces (such as scraping) (Lloyd et al., J. Cell Sci. 22: 671-684 (1976); Whur et al., J. Cell Sci. 23: 193-209 (1977); Freyer and Sutherland, Cancer Res. 40: 3956-3965 (1980); Lydersen et al., Bio/Technol. 1: 63-67 (1985)). The dispersion of aggregates was also tested with DNAse (Jordan et al. Animal Cell Technology: Developments, Processes and Products, eds: Spier et al., 418-420 (1992), pub: Butterworth-Heinemann, Oxford; Renner et al., Biotechnol. Bioeng., 41: 188-193 (1993),) or with hypo-osmolar medium (Leibovitz et al., Int. J. Cell Cloning, 1: 478-485 (1983)). All of these treatments are usually insufficient individually to obtain a uniformly dispersal of viable individual cells. There usually remain some cell clusters visible with the microscope and/or to the naked eye. A cell aggregate or cluster is a mass of variable size, sometimes visible by the naked eye, formed by the union of individual cells together or by the union of cells to at least one other material (i.e. debris, extracellular matrix) present in the initial cell suspension. By definition, a cell aggregate has a minimal size of about 800 μm, in particular a minimal size of about 600 μm, particularly of about 400 μm, preferably of about 200 μm, more preferably of about 100 μm.
Dislodged and dispersed cell suspensions may also need to undergo several downstream treatments, for example to remove chemical compounds used during cell harvest, such as trypsin. These steps are time consuming and increase the cost of the product and may result in undesirable reaggregation. For example, a centrifugation step may be performed to remove undesired chemical compounds. This process, however, leads to the formation of a supernatant containing the chemical compounds and which will be discarded, and a pellet comprising cells to be harvested. When compacted into a pellet, the cells are so close and pushed together that cell aggregates are formed.
Compared to microorganisms such as viruses and bacteria, eukaryotic cells, and especially animal cells, are very fragile and shear sensitive due to the lack of a durable cell wall. Shear sensitivity is also related to the cell type (i.e. whether they are fibroblasts, lung cells, kidney cells, etc.), the culture age and history (old cultures having a high number of passages contain more fragile cells) and maintenance conditions (variations of the culture conditions, such as temperature, osmotic pressure, etc generate stresses). Virus infection may also lead to an increase of the shear sensitivity of infected cells.
In mouse and human cell culture experiments, wall shear stresses of 100 N/m2 over 0.5 seconds residence time cause a significant cell death rate. Studies on embryonic kidney cells showed that shear stresses of less than 0.26 N/m2 caused a slight reduction in viability and no change in cell morphology (Harbour et al., Adv. Biochem. Eng., Vol. 29. pub: Springer-Verlag (New York), (1984)).
As a general consideration, therefore, shear forces applied on a cell suspension could result in a decrease in cell viability. Shearing forces may decrease the yield of the viable cells and can also reduce the ability of the cells to divide by inhibition of cell mitosis.
Since for pharmaceutical use good cell viability is preferred, a gentle method of dispersing a cell suspension containing cell aggregates is needed. The technology used has to be efficient to release individual cells in high production yields, but has also to be gentle enough to avoid significant reduction in viability.
Known cell culture manipulation methods may involve dispersion with gentle methods, for example with gentle pipetting (ECACC Handbook, Fundamental Techniques in Cell Culture. A Laboratory Handbook, “Protocol 5—Subculture of suspension cell lines”, 2005, edited by Sigma-Aldrich). Pipetting is typically performed manually by repeated aspiration and rejection of the cell suspension until cell clusters have all disappeared. This manual operation is not, however, consistent and reproducible. Different results can be seen using the same cell culture starting material from one pipette to another, or one operator to another. In addition, shear damage is a function of both shearing time and shearing forces. Pipetting too vigorous and/or over too long a period can damage the cells and result in low viability. Alternatively, pipetting too gently or inconsistently and difficulty of determining when cell clusters have disappeared can result in a poor cell yield because remaining cell aggregates will be discarded during subsequent filtration steps. Beside this lack of robustness, the gentle pipetting technique is tedious and requires open phases that increase the risks of contamination. Pipetting is not amenable to large volume processing.
Accordingly, there is still a need for large-scale processes for the dispersion of cell aggregates, and preferably done in a closed system to avoid contamination risks. The present invention addresses these problems by providing a flow-through dispersion device for dispersion of shear sensitive aggregates, notably culture suspensions containing cell aggregates, while respecting the integrity of the individual cells and flow-through methods for dispersing shear-sensitive cell aggregates to release individual cells.